VNN1 promotes atherosclerosis progression in apoE-/- mice fed a high-fat/high-cholesterol diet

Abstract

Accumulated evidence shows that vanin-1 (VNN1) plays a key part in glucose metabolism. We explored the effect of VNN1 on cholesterol metabolism, inflammation, apoptosis in vitro, and progression of atherosclerotic plaques in apoE(-/-) mice. Oxidized LDL (Ox-LDL) significantly induced VNN1 expression through an ERK1/2/cyclooxygenase-2/PPARα signaling pathway. VNN1 significantly increased cellular cholesterol content and decreased apoAI and HDL-cholesterol (HDL-C)-mediated efflux by 25.16% and 23.13%, respectively, in THP-1 macrophage-derived foam cells (P < 0.05). In addition, VNN1 attenuated Ox-LDL-induced apoptosis through upregulation of expression of p53 by 59.15% and downregulation of expression of B-cell lymphoma-2 127.13% in THP-1 macrophage (P < 0.05). In vivo, apoE(-/-) mice were divided randomly into two groups and transduced with lentivirus (LV)-Mock or LV-VNN1 for 12 weeks. VNN1-treated mice showed increased liver lipid content and plasma levels of TG (124.48%), LDL-cholesterol (119.64%), TNF-α (148.74%), interleukin (IL)-1β (131.81%), and IL-6 (156.51%), whereas plasma levels of HDL-C (25.75%) were decreased significantly (P < 0.05). Consistent with these data, development of atherosclerotic lesions was increased significantly upon infection of apoE(-/-) mice with LV-VNN1. These observations suggest that VNN1 may be a promising therapeutic candidate against atherosclerosis.

Keywords: apolipoprotein E; inflammation; oxidized low density lipoprotein; vanin-1.

Fig. 1.

Effect and mechanism of Ox-LDL on VNN1 expression in THP-1 macrophages. A–D: Effects of Ox-LDL on VNN1 expression in THP-1 macrophages. A, C: THP-1 macrophages were treated with Ox-LDL at 0, 25, 50, and 100 μg/ml for 48 h, respectively. B, D: THP-1 macrophages were treated with 50 μg/ml of Ox-LDL for 0, 12, 24, and 48 h, respectively. Levels of VNN1 gene and protein were measured by real-time qPCR and Western blotting, respectively. E: Levels of VNN1 protein in human tissues of normal arterial intima (n = 5, 3 men; age 32.5 ± 7.8 years) and advanced atherosclerotic plaques (n = 5, 2 men; age 63.7 ± 8.9 years) were analyzed by Western blotting. F: THP-1 macrophages were transfected with control or ERK1/2 siRNA, COX-2 siRNA, or PPARα siRNA, and then incubated with 50 μg/ml Ox-LDL for 48 h. Protein expression was measured by Western blotting. G: THP-1 macrophages were transfected with control or ERK1/2 inhibitor PD98059, COX-2 inhibitor NS-398, or PPARα inhibitor GW6471, and then incubated with 50 μg/ml Ox-LDL for 48 h. Protein expression was measured by Western blotting. Data are the mean ± SD of five independent experiments, each done in triplicate. * P < 0.05 versus control.

Fig. 2.

Effect of VNN1 on lipid loading, lipid content, and cholesterol efflux. THP-1 macrophages were transduced with LV-Mock or LV-VNN1. A: Then cells were incubated with 5 μg/ml of Dil-labeled Ox-LDL for 1 h and uptake of Dil-labeled Ox-LDL analyzed by flow cytometry. B, C: Cells were labeled with 0.2 μCi/ml [³H]cholesterol and cholesterol-loaded using 50 μg/ml Ox-LDL. The percentage of cholesterol efflux to apoAI (B) and HDL (C) was analyzed with a liquid scintillation counting assay. D: HPLC was done to determine the TC, free cholesterol (FC), and cholesteryl ester (CE) content in cells. E: Protein levels were measured by Western blot analyses. Data are the mean ± SD from five independent experiments, each carried out in triplicate. * P < 0.05 versus control group.

Fig. 3.

Effect and mechanism of VNN1 on apoptosis. A: THP-1 macrophages and VSMCs were transduced with LV-Mock or LV-VNN1, respectively. B: THP-1 macrophages and VSMCs were transduced with LV-Mock or LV-VNN1 and then treated with 100 μg/ml of Ox-LDL for 24 h, respectively. A, B: The proportion of apoptotic cells was assessed by flow cytometry. C, D: THP-1 macrophage were transduced with LV-Mock or LV-VNN1 and then treated with or without 100 μg/ml of Ox-LDL for 24 h as indicated. Protein levels were measured by Western blot analyses. Data are the mean ± SD from five independent experiments, each undertaken in triplicate. * P < 0.05 versus control group.

Fig. 4.

Effect of VNN1 on RCT and lipid deposition in the liver. A: C57BL/6 mice were treated with an HFD or a chow diet. VNN1 protein expression levels in various tissues were measured by Western blot. B: The expression levels of VNN1 in the LV-treated mice were measured by Western blot. C: After 12 weeks of the treatment indicated, apoE^−/− mice were injected subcutaneously with [³H]cholesterol-labeled Ac-LDL-loaded BMDMs. Data are the percentage of the [³H]cholesterol tracer relative to that of total counts per minute tracer injected ± SD; n = 10, * P < 0.05 versus control group. (a) Time course of [³H]cholesterol distribution in plasma. (b) Hepatic levels of [³H]cholesterol tracer after 48 h. (c) Fecal levels of [³H]cholesterol tracer. Feces were collected continuously from 0 h to 48 h postinjection. D: (a) Liver cryosections were stained with Oil Red O. Original magnification: ×100. (b) Hepatic content of TG and TC in apoE^−/− mice was assessed by enzymatic analyses. n = 10, * P < 0.05 versus control group. E: THP-1 macrophage-derived foam cells were treated with LV-Mock or LV-VNN1, respectively, and then levels of inflammatory cytokines in the medium measured with ELISA. Data are the mean ± SD from five independent experiments, each done in triplicate. * P < 0.05 versus control group.

Fig. 5.

Effect of VNN1 on the initiation and development of atherosclerosis in apoE^−/− mice. A: (a) Representative staining of en face aorta with Oil Red O. (b) En face lesions were analyzed in apoE^−/− mice. B: (a) Representative staining of aortic valves with Oil Red O. (b) Total lesions in the aortic valves of apoE^−/− mice were analyzed. C: (a) Cryosections of aortic valves from apoE^−/− mice were stained by IHC means for the macrophage marker CD68. (b) The integral optical density of CD68 in aortic-valve cryosections from apoE^−/− mice was analyzed. D: (a) Representative image of aortic valves with TUNEL staining. (b) Total apoptosis in the aortic valves of apoE^−/− mice was analyzed. E: Levels of protein expression in the aortic tissues of apoE^−/− mice were analyzed by Western blotting. F: Levels of protein expression in peritoneal macrophages derived from apoE^−/− mice were analyzed by Western blot (n = 5 mice/group). Data are the mean ± SD; n = 10. All experiments were done in triplicate, except as indicated. * P < 0.05 versus control group.

Fig. 6.

VNN1 expression and function can be regulated by Ox-LDL to promote atherosclerosis. These harmful effects are accompanied by decrease of cholesterol efflux, promotion of inflammatory molecule expression, and decrease of macrophage apoptosis, thus promoting atherosclerotic plaque formation. Ox-LDL upregulates VNN1 expression through ERK1/2/COX-2/PPARα signaling pathway. Then, VNN1 could inhibit cellular cholesterol efflux through reducing expression of ABCA1, ABCG1, SR-BI, and NPC1 by inhibiting expression of PPARγ and LXRα. In addition, VNN1 could promote inflammation through inducing expression of TNF-α, IL-1β, IL-6, ICAM-1, and VCAM-1. Moreover, VNN1 might reduce macrophage apoptosis through inhibiting P53 expression and inducing BCL-2 expression.